Bone graft substitutes (BGS) are commonly used as a less-invasive alternative to autograft for the repair of osseous defects. Autograft, however, remains the gold standard in grafting applications due to its supply of a structural scaffold, osteoinductive growth factors, and osteogenic cells. A promising surgical option to autograft is the use of a composite graft consisting of a BGS combined with bioactive (i.e, osteoinductive and osteogenic) elements. Examples of bioactive elements include platelets (which secrete osteoinductive growth factors) and stem cells (which differentiate into osteogenic bone-forming cells). Sources of these bioactive elements include bone marrow aspirate and blood, which are less invasive to procure from a patient than autograft. With this in mind, techniques have been developed to procure and concentrate bioactive elements from bone marrow aspirate and blood for use in tissue healing applications. Centrifugation is typically used to separate osteogenic/osteoinductive cells from other blood constituents based on differences in density. However, preparation of cell-rich suspensions can be arduous in addition to requiring the purchase and maintenance of a centrifuge in the operating room. Accordingly, a point-of-care surgical technique that could concentrate stem cells and platelets from blood or bone marrow aspirate without the need for centrifugation is desirable. This would give a surgeon a convenient method to rapidly prepare a bioactive BGS that would be a viable and less invasive alternative to autograft.
In Connolly et al. (1989), marrow extracted from rabbits was concentrated by simple centrifugation, isopyknic centrifugation, and gravity sedimentation, all of which increased the nucleated cell count on average compared to whole marrow. The osteogenic effect of bone marrow was tested in rabbits, using a chamber that had been implanted in a peritoneal cavity (ectopic site) and in a delayed-union model (orthotopic site). Osteogenesis was accelerated in both sites after concentration of marrow elements after centrifugation, but not after gravity sedimentation. Although a method to increase the nucleated cell density within bone marrow aspirate was described, centrifugation was utilized to concentrate stem cells in marrow. Furthermore, the concentrate was not used in conjunction with an osteoconductive scaffold material.
PCT Patent Application WO 96/27397 provides a plasma-buffy coat concentrate that comprises plasma, platelets, and fibrinogen. When the concentrate is combined with a fibrinogen activator in sufficient concentration to initiate clot formation, a wound sealant is formed. Also provided is a method for processing blood to produce the plasma-buffy coat concentrate. The method comprises centrifuging anticoagulated blood to remove red blood cells and to produce a plasma-buffy coat mixture. Water is removed from the mixture by hemofiltration to produce the plasma-buffy coat concentrate. A fibrinogen activator is mixed with the plasma-buffy coat concentrate to produce a wound sealant that can be used for multiple clinical indications, including bone-grafting applications.
U.S. Pat. Nos. 6,010,627 and 6,342,157 provide a device and a method for concentrating a blood fraction, typically plasma, to provide a concentration of blood procoagulant proteins, such as fibrinogen, and cellular components, such as platelets, white blood cells, or buffy coat cells. Water is removed from plasma by hemofiltration to produce a plasma-buffy coat concentrate. The resultant concentrate is suitable for use in the preparation of coagulum-based wound sealants. The method utilizes a hemofilter, which is a type of hollow fiber filter used for blood processing, in order to concentrate cells and proteins within a blood fraction. The prior art defines an ultrafiltration unit having a semi-permeable membrane with a molecular weight cut-off of about 30,000 daltons, which allows for the concentration of fibrinogen protein, useful for coagulation upon addition of a fibrinogen activator to the cell/protein concentrate. However, U.S. Pat. Nos. 6,010,627 and 6,342,157 do not describe the fiber filter for the concentration of mesenchymal stem cells and platelets from either whole blood or bone marrow aspirate. Accordingly, a larger cut-off filter (i.e., exceeding 30,000 daltons) may be used in the present invention to concentrate cells since the recovery of proteins is not critical. Because larger cut-off filters have higher flow rates and require lower operating pressure than smaller cut-off filters, the filters in the present invention will require less time and force to operate compared to the filters disclosed therein.
Muschler et al. (2002; U.S. Pat. Nos. 5,824,084 and 6,049,026) provide a method for preparing a composite BGS in which bone marrow aspirate is passed through a porous substrate. The osteoprogenitor cells are selectively retained in the substrate, resulting in a composite graft that contains an enriched (i.e., greater) number of progenitor cells compared to an equivalent volume of bone marrow aspirate. A method is provided for passing marrow through an implantable porous scaffold that acts as an affinity column for mesenchymal stem cells. The stem cells are selectively retained in the substrate, resulting in a composite graft that contains an enriched (i.e., greater) number of stem cells compared to an equivalent volume of bone marrow aspirate. However, the disclosure of Muschler does not describe methods and compositions for the enrichment of platelets.
PCT Patent Application WO 00/61256 and U.S. Pat. No. 6,398,972 describe an automated method and apparatus for producing platelet-rich plasma or a platelet concentrate from a physiological solution, preferably blood. Processing is carried out by a centrifuge that receives a disposable container having two chambers. Whole blood is placed into one of the two chambers, and the centrifuge is then operated to cause the red blood cells to sediment to the bottom of one chamber, resulting in a supernatant of platelet-rich plasma. The centrifugation is stopped, which causes the platelet-rich plasma to drain to the second chamber. The platelet-rich plasma in the second chamber is then centrifuged a second time by restarting the centrifuge. The centrifuge is stopped, resulting in: (1) red blood cells in one chamber; (2) platelet concentrate at the bottom of the second chamber; and (3) platelet poor plasma as the supernatant in the second chamber. A portion of the platelet poor plasma is removed from the second chamber, leaving a remaining portion of the platelet poor plasma and platelet concentrate in the second chamber. The remaining platelet concentrate is suspended in the platelet-poor plasma remaining in the second chamber to obtain platelet rich plasma.
Thus, the present invention as described herein provides a need in the art of clinical therapy, particularly bone therapy, lacking in the present methods.